A diverse range of assays are used in research, analysis, development and clinically to detect analytes of interest. Immunoassays are a particularly useful form of assay that exploit the specificity, strength and diversity of antibody-antigen reactions to analyse samples and detect specific components therein. A wide range of immunoassay techniques are available, such as those described in Wild D. “The Immunoassay Handbook” Nature Publishing Group, 2001.
The detection of antibodies to specific antigens has been used in the diagnosis of specific disease states. For example, the presence of antibody to hepatitis A virus indicates infection with hepatitis A virus and the likelihood of immunity to subsequent infection with that virus. The detection of different class of antibody or immunoglobulin can also provide further information about a disease or a subject's immune status. For example, a current disease state may be distinguished by the presence of IgM antibody while infection in the more distant past may be distinguished by the detection of IgG antibodies.
Methods for the detection of antibody to specific antigens are also known. For example, the enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) are routinely used in laboratories. These methods generally require some level of skill in laboratory techniques. A variety of methods have also been developed which require little skill and are rapid to perform, and which are therefore suitable for the detection of antibody to specific antigens at the point of care or analysis. In particular, immunochromatographic or dipstick enzyme-linked immunosorbent kits have been developed to assay for a number of infections including viral infections.
In many immunoassays, it is necessary to form a conjugate containing the specific antigen together with a detectable marker. The antigen of a virus may, for example, be conjugated with colloidal gold such that immune reactivity between the antigen-colloidal gold complex and specific antibody in a device can be detected. Alternatively, the antigen of a virus may be conjugated with an enzyme such as horseradish peroxidase, such that immune reactivity between the antigen-enzyme complex and specific antibody can be detected in an ELISA.
However, the process of conjugation between colloidal gold or enzyme and the antigen of interest may result in a reduction of the immune reactivity between the antigen and the antibody which it is intended to detect. Specifically, the antibody binding site may be the physical site of binding to the colloidal gold or enzyme such that it is inaccessible to the antibody molecule, or the process of binding may alter the conformation of the antigen such that it is no longer recognised by the antibody molecule. At the least, binding of the antigen to colloidal gold or enzyme may be in a random orientation, such that only a proportion of the antigen molecules are available to react with patient antibody to give a detectable signal in a diagnostic test.
The preparation of gold or enzyme conjugates with antigen requires the use of highly purified antigens to prevent the formation of gold or enzyme conjugates containing contaminating proteins which could then react with antibody resulting in non-specific reactions and unreliable test results. The processes used for extensive purification of antigens add to the cost of such preparations, and may also result in a reduction of immune reactivity of the antigen.
Viral particles, albeit with some notable exceptions, are typically roughly spherical and about 30 to 100 nm in diameter. Viral Hepatitis may be caused by one or more viruses such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV) and hepatitis non A-E virus (HNAEV). Chronic liver disease is frequently caused by Hepatitis B, C or D. HAV is a highly contagious virus which infects the liver and causes flu-like symptoms and liver damage. It is the most common form of hepatitis being transmitted through ingestion of contaminated faeces and currently is thought to have infected at least 2 billion people or 40 million people across the world each year, assuming they are infected over a 50 year period. HAV is diagnosed in the laboratory setting by detecting IgM antibodies to antigens from HAV.
Despite the existence of a wide range of immunoassays for detecting patient antibodies to specific antigens, the practical limitations of adapting these assays to a format suitable for use with viral antigens and antibodies determined thereby in the general population and to a format suitable for use in environments without access to laboratory equipment or suitably trained operators has hitherto prevented the successful development of a diagnostic kit for hepatitis A virus which provides robust, rapid and accurate diagnosis of infection even away from the laboratory or clinic.